Monoclonal antibodies against the antigen-specific T cell receptor (TCR)/CD3-complex are able to efficiently activate T cells. This activation, however, requires the antibody to be—via its Fc portion—multimerized on the surface of Fc receptor expressing cells, which often also provide accessory signals for T cell activation (Davis, L., Vida, R. and Lipsky, P. E., Regulation of human T lymphocyte mitogenesis by antibodies to CD3, J. Immunol. [1986] 137: 3758-3767).
Bispecific antibodies, which recognize both an antigen on target cells (e.g. FLT3 or CD19 on leukemia cells, the CSPG4-antigen on melanoma cells or EGFR on glioblastoma cells) and the antigen specific T cell receptor (TCR)/CD3-complex, are likewise able to activate T cells (Jung, G., Ledbetter, J. A., and Muller-Eberhard, H. J., Induction of cytotoxicity in resting human T lymphocytes bound to tumor cells by antibody heteroconjugates, Proc. Natl. Acad. Sci. U.S.A [1987] 84: 4611-4615; Jung, G., & Eberhard, H. J., An in-vitro model for tumor immunotherapy with antibody heteroconjugates, Immunol. Today [1988] 9: 257-260; Jung, G., Brandl, M., Eisner, W., Fraunberger, P., Reifenberger, G., Schlegel, U., Wiestler, O. D., Reulen, H. J., Wilmanns, W. Local immunotherapy of glioma patients with a combination of 2 bispecific antibody fragments and resting autologous lymphocytes: evidence for in situ T-cell activation and therapeutic efficacy, Int J Cancer. [2001] 91: 225-30), and in addition to focus the activated cells on the target cell (Staerz, U. D., Kanagawa, O., and Bevan, M. J., Hybrid antibodies can target sites for attack by T cells, Nature [1985] 314: 628-631; Perez, P., Hoffman, R. W., Shaw, S., Bluestone, J. A., and Segal, D. M. Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody, Nature [1985] 316: 354-356; Jung, G., Honsik, C. J., Reisfeld, R. A. and Muller-Eberhard, H. J. Activation of human peripheral blood mononuclear cells by anti-T3: killing of tumor target cells coated with anti-target-anti-T3 conjugates, Proc. Natl. Acad. Sci. U.S.A, 83: 4479-4483, 1986). As a result T cell mediated lysis of tumour cells occurs. Agonistic antibodies to T-cell costimulatory molecule such as CD28, enhance anti-CD3 mediated T-cell activation. Such costimulatory antibodies are particularly effective if they are also provided in a bispecific format (Grosse-Hovest, L., Hartlapp, I., Marwan, W., Brem, G., Rammensee, H. G., and Jung, G., A recombinant bispecific single-chain antibody induces targeted, supra-agonistic CD28-stimulation and tumor cell killing, Eur. J. Immunol. [2003] 33: 1334-1340). In any case, we regard it as an absolute requirement for therapeutic applications of bispecific antibodies having CD3 specificity that binding to Fc receptors can be excluded (Jung, G., and Eberhard, H. J., An in-vitro model for tumor immunotherapy with antibody heteroconjugates, Immunol. Today [1988] 9: 257-260; Jung, G., Freimann, U., Von Marshall, Z., Reisfeld, R. A., and Wilmanns, W., Target cell-induced T cell activation with bi- and trispecific antibody fragments, Eur. J. Immunol. [1991] 21: 2431-2435). Such binding to Fc receptors would result in T cell activation in vivo, which occurs, regardless of the binding to a target antigen, at any location where Fc receptor expressing cells can be found, for instance within the entire hematopoietic, lymphatic and reticulo-endothelial system. According to experience such T cell activation results in systemic activation of T cells, accompanied by a cytokine release syndrome, a dreaded adverse reaction during therapeutic use of T cell activating cytokines or antibodies (Rosenberg, S. A., Lotze, M. T., Yang, J. C., Aebersold, P. M., Linehan, W. M., Seipp, C. A., and White, D. E., Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients, Ann. Surg. [1989] 210: 474-484; Tibben, J. G., Boerman, O. C., Massuger, L. F., Schijf, C. P., Claessens, R. A., and Corstens, F. H., Pharmacokinetics, biodistribution and biological effects of intravenously administered bispecific monoclonal antibody OC/TR F(ab′)2 in ovarian carcinoma patients, Int. J. Cancer [1996] 66: 477-483; Kroesen, B. J., Buter, J., Sleijfer, D. T., Janssen, R. A., van der Graaf, W. T., The, T. H., de, L. L. and Mulder, N. H., Phase I study of intravenously applied bispecific antibody in renal cell cancer patients receiving subcutaneous interleukin 2, Br. J. Cancer [1994] 70: 652-661). Hence, the aim in formatting bispecific CD3 antibodies needs to be avoiding an Fc mediated systemic activation of T cells, and thereby allowing target cell restricted activation, which is exclusively dependent on binding of the target portion of the bispecific antibody to the corresponding target antigen (Jung, G., & Eberhard, H. J., An in-vitro model for tumor immunotherapy with antibody heteroconjugates, Immunol. Today [1988] 9: 257-260; Jung, G., Freimann, U., Von Marshall, Z., Reisfeld, R. A., and Wilmanns, W. Target cell-induced T cell activation with bi- and trispecific antibody fragments, Eur. J. Immunol. [1991] 21: 2431-2435). From the above said it emerges that when selecting the target antigen, expression as restricted to malign cells as possible has to be taken care of. In this way activation by non-malign cells and an accompanying release of cytokines can be kept as low as possible.
Similar considerations apply if bispecific antibodies are constructed that contain agonistic effector antibodies binding to triggering receptors on immune cells other than T cells, such as CD16 expressed on NK cells. In any case, Fc-mediated binding of the antibodies to Fc receptors should be avoided according to the reasoning outlined above for T cells.
The bispecific antibody which has proceeded furthest in clinical development today is Blinatumomab (Micromet, Inc., Rockville, Md.), a bispecific single chain antibody with CD19×CD3 specificity and a remarkable therapeutic activity against lymphoma and leukemia cells (Bargou, R., et al., Tumor regression in cancer patients by very low doses of a T cell-engaging antibody, Science [2008] 321: 974-977; Topp, M. S., et al., Targeted therapy with the T-cell-engaging antibody blinatumomab of chemotherapy-refractory minimal residual disease in B-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival, J. Clin. Oncol. [2011] 29: 2493-2498).
Since the single chain format does not contain any domain of the Fc part, this antibody is target cell restricted within the above explained meaning, i.e. it only activates T cells in the presence of CD19 expressing target cells (Brischwein, K., et al., Strictly target cell-dependent activation of T cells by bispecific single-chain antibody constructs of the BiTE class, J. Immunother. [2007] 30: 798-807).
CD19 is, however, also expressed on normal B cells so that, despite target cell restriction, following therapeutic application, a systemic release of cytokines occurs, causing significant cytotoxicity already at daily doses around 100 μg (Bargou, R., et al., Tumor regression in cancer patients by very low doses of a T cell-engaging antibody, Science [2008] 321: 974-977; Topp, M. S., et al., Targeted therapy with the T-cell-engaging antibody blinatumomab of chemotherapy-refractory minimal residual disease in B-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival, J. Clin. Oncol. [2011] 29: 2493-2498).
In addition the single chain format has the following disadvantages: (i) the molecular weight of about 50 kDa is relatively low and is associated with a short serum half life, (ii) antibodies of this format easily aggregate and (iii) are difficult to produce in conventional fermenting processes (Grosse-Hovest, L., Hartlapp, I., Marwan, W., Brem, G., Rammensee, H. G., and Jung, G., A recombinant bispecific single-chain antibody induces targeted, supra-agonistic CD28-stimulation and tumor cell killing, Eur. J. Immunol. [2003] 33: 1334-1340; Grosse-Hovest, L., et al., Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing, Proc. Natl. Acad. Sci. U.S.A [2004] 101: 6858-6863).
It is therefore an object of the present invention to provide a bispecific antibody molecule that overcomes at least some of the above difficulties and that can be generally used in therapy, amongst others for strictly target cell restricted activation of immune cells as described above.